The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.
Melanoma and Basal Cell Carcinoma
Skin cancer is the most common of all cancers, afflicting more than one million Americans each year, a number that is rising rapidly. It is also the easiest to cure, if diagnosed and treated early. If allowed to progress to the point where it spreads to other sites, the prognosis is very poor. More than 8,000 melanoma deaths now occur per year.
Melanoma is a malignant tumor of melanocytes. Melanocytes predominantly occur in skin, between the outer layer of the skin (the epidermis) and the next layer (the dermis), but are also found in other parts of the body, including the bowel and the eye (see uveal melanoma). Melanoma can occur in any part of the body that contains melanocytes or as a metastatic tumor of unknown primary lesion. Melanoma is less common than other skin cancers but is much more dangerous and causes the majority (75%) of deaths related to skin cancer.
Melanoma arises from DNA damage to melanocytes. The early stage of the disease commonly begins with a radial growth phase when the tumor is confined to the epidermis followed by a dermal “vertical growth phase” (VGP). Some melanomas attain further invasive potential, growing into the surrounding tissue and may spread around the body through blood or lymph vessels to form metastases.
An immunological reaction against the tumor during the VGP may be judged by the presence and activity of the tumor infiltrating lymphocytes (TILs). These cells sometimes attack the primary tumor, and in certain cases, the primary tumor regresses with diagnosis of only the metastatic tumor.
Multiple genetic events have been related to the pathogenesis (disease development) of melanoma. Some cases of melanoma have a clear genetic predisposition. Germline mutations in CDKN2A, CDK4, MC1R, MDM2 SNP309 and in genes associated with xeroderma pigmentosum (XP) predispose patients to developing melanoma. Other cases of familial melanoma are genetically heterogeneous, and putative loci for familial melanoma have been identified on the chromosome arms 1p, 9p and 12q.
Clinical and Pathological Diagnosis
Melanoma is usually first detected by visual examination of pigmented lesions of the skin, notably those that show: (A) asymmetry, (B) a border that is uneven, ragged, or notched, (C) coloring of different shades of brown, black, or tan and (D) diameter that has recently changed in size. In contrast, non-neoplastic moles or nevi are symmetrical, have a regular border, even coloration, and show no change in size/diameter over time. The main diagnostic concern is in distinguishing between a benign nevus, a dysplastic nevus-which may show progression over time, and a melanoma. Moles that are irregular in color or shape undergo further workup for melanoma. Following a visual examination and a dermatoscopic exam, or in vivo diagnostic tools such as a confocal microscope, a sample (biopsy) of the suspicious mole is usually obtained.
Sample Preparation
When an atypical mole has been identified, a skin biopsy takes place in order to best diagnose it. Local anesthetic is used to numb the area, then the mole is biopsied. The biopsy material is then sent to a laboratory to be evaluated by a pathologist. A skin biopsy can be a punch or shave biopsy, or complete excision. The complete excision is the preferred method, but a punch biopsy can suffice if the patient has cosmetic concerns (i.e. the patient does not want a scar) and the lesion is small. A scoop or deep shave biopsy is generally avoided due to risk of transecting a melanoma and thereby losing important prognostic information.
Most dermatologists and dermatopathologists use a diagnostic schema for classifying melanocytic lesions based on how symmetrical the lesion is and the degree of cytologic atypia in the melanocytes. In this classification, a nevus is classified as unequivocally benign, atypical/dysplastic, or clearly melanoma. A benign nevus exhibits no significant cytologic atypia and symmetrical growth. An atypical mole is read as having either asymmetrical growth, and/or having (mild, moderate, or severe) cytologic atypia. Usually, cytologic atypia is of more important clinical concern than architectural atypia. Along with melanoma, nevi with moderate to severe cytologic atypia may require further excision to make sure that the surgical margin is completely clear of the lesion.
Important aspects of the skin biopsy report for melanoma, including the pattern (presence/absence of an in situ component, radial or vertical growth), depth of invasion, presence of lymphocyte infiltrate, presence/absence of vascular or lymphatic invasion, presence/absence of a preexisting benign melanoma and the mitotic index. A further important aspect of the skin biopsy report for atypical nevi and melanoma is for the pathologist to indicate if the excision margin is clear of tumor. If there is any atypical melanocytes at the margin or if a melanoma is diagnosed, a reexcision is performed. Lymph node dissection may also be performed based on the tumor parameters seen on the initial biopsy and on the reexcision.
Further molecular testing may be performed on melanoma biopsies, reexcision or lymph node metastatic samples to assess for targetable genetic changes to help select optimal therapy.
BRAF
BRAF is a human gene that makes a protein called B-Raf. The gene is also referred to as proto-oncogene B-Raf and v-Raf murine sarcoma viral oncogene homolog B1, while the protein is more formally known as serine/threonine-protein kinase B-Raf. B-Raf is a member of the Raf kinase family of serine/threonine-specific protein kinases. This protein plays a role in regulating the MAP kinase/ERK signaling pathway, which affects cell division, differentiation, and growth factor expression.
In 2002, BRAF was shown to be mutated in human cancers. More than 30 mutations of the BRAF gene associated with human cancers have been identified. The frequency of BRAF mutations varies widely in human cancers from approximately 60% of melanomas and some types of benign nevi, to approximately 1-10% of common carcinomas such as lung adenocarcinoma (ACA) and colorectal cancer. In 90% of BRAF-mutated tumors, thymine is substituted for adenine at nucleotide 1799. This leads to valine (V) being substituted for by glutamate (E) at codon 600 (V600E) in the activation segment. This mutation has been widely observed in papillary thyroid carcinoma, colorectal cancer, melanoma and non-small-cell lung cancer. In June 2011, a team of Italian scientists used massively parallel sequencing to pinpoint mutation V600E as a likely driver mutation in 100% of cases of hairy cell leukemia. Less commonly, V600E mutation can also occur by a double nucleotide substitution.
BRAF mutations which have been found are R462I, I463 S, G464E, G464V, G466A, G466E, G466V, G469A, G469E, N581S, E586K, D594V, F595L, G596R, L597V, T599I, V600D, V600E, V600K, V600R, K601E, E602K and A728V, etc. Most of these mutations are clustered in two regions of the gene: the glycine-rich P loop of the N lobe and the activation segment and flanking regions. Many of these mutations change the activation segment from an inactive state to an active state. For example in V600 mutations, the aliphatic side chain of Val600 interacts with the phenyl ring of Phe467 in the P loop. Replacing the medium-sized hydrophobic Val side chain with a larger and charged residue (such as the Val to Glu, Asp, Lys, or Arg changes seen in human tumors) can destabilize the interactions that maintain the DFG motif in an inactive conformation, resulting in conformational shift in the active position. Each BRAF kinase mutation has a variable effect on MEK phosphorylation activity, with most mutations having higher phosphorylation activity than the unmutated B-Raf protein, but some mutations show reduced or even absent kinase activity, termed “inhibitory” BRAF mutations. The effect of these inhibitory mutations appears to be to activate wild-type C-Raf, which then signals to ERK.
BRAF has also emerged as important drug target for tumor therapy. Drugs that treat cancers driven by BRAF mutations have been developed. On Aug. 17, 2011, one of them, vemurafenib, was approved by FDA for treatment of advanced-stage melanoma. Other BRAF-directed kinase inhibitors include GDC-0879, PLX-4720, sorafenib tosylate. dabrafenib, and LGX818.
DDR2
Discoidin domain receptor family, member 2, also known as DDR2 or CD 167b (cluster of differentiation 167b), is a receptor tyrosine kinase (RTK) that regulates cell growth, differentiation, and metabolism in response to extracellular signals. DDR2 mutation has been previously reported in 3-4% of squamous cell carcinoma (SCC) of the lung. In lung SCC, a few cases with DDR2 mutation were shown to have clinical response to treatment with the tyrosine kinase inhibitor dasatinib (Cancer Discov. 2011 Apr. 3; 1(1): 78-89). The data suggested that DDR2 may be an important therapeutic target in SCC.
DDR2 protein comprises an extracellular discoidin (DS) domain, a transmembrane domain and a kinase domain. The kinase domain is located at amino acids 563 to 849 of the full length protein (which includes the signal peptide) and the DS domain is located at amino acids 22-399. The nucleotide sequence of human DDR2 mRNA variant 2 is shown in GenBank Accession no. NM_006182.